Use Of Diagnostic Techniques Research Articles

1277-P: Spatial Whole Transcriptome Profiling of the Human Pancreas Uncovers Unique Insights into Histological Structures

Understanding the physiology of the pancreas and pathology that occurs during diabetes requires knowing transcriptional patterns driving biological activity within the functional structures of the tissue. Previous technologies have not enabled spatially resolved high plex profiling to elucidate these biological pathways. Using the unique ability of GeoMx® Digital Spatial Profiler to resolve functional units within FFPE tissue in situ, we provide whole transcriptome expression data from islets, acini, and ducts from four pancreata of nondiabetes patients. On average, 6000-8000 genes were detected from functional groups within each histological structure. Data were validated against the independent Human Protein Atlas, with ∼90% concordance between RNA expression and known protein localization. Deconvolution based on single-cell RNASeq enabled calculation of the proportion of distinct cell types within each structure, confirming cell composition. For example, while ductal components consist of small islands punctuating the acini, over 3000 genes were identified to be highly specific to each structure (FDR < 0.05) , and enriched for pathways related to focal adhesion, ECM structure, and diabetic disease processes. Within islets, we identified >300 genes with differences between α- and β-cells (FDR < 0.2) . Functional characterization confirmed enrichment of insulin biosynthesis and secretion pathways, with genes such as insulin, glucagon, and IAPP expression captured accurately with a single assay. Determination of expression in four individual pancreas samples identified variation between tissues, which may be central to health and disease processes. In conclusion, whole transcriptome data for spatial gene expression profiles of structures of the non-diseased pancreas are publicly available and can be used as standards to inform future profiling studies in normal and diseased tissue. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Disclosure W.Yang: None. M.F.Hogan: Employee; NanoString. Y.Liang: None. J.M.Beechem: None.

Safety and effectiveness of vascular closure devices in interventional radiological procedures.

Although it is well known that vascular closure devices (VCD) are commonly used in therapeutic interventional radiological procedures, standard use in diagnostic procedures is not as well studied. The aim of this study was to determine the real-world safety and effectiveness of the VCD in both diagnostic and therapeutic interventional radiological procedures. A retrospective, single center study included all patients where VCDs were used for either a diagnostic or therapeutic interventional procedure. Various demographic and clinical risk factors were recorded and examined for any significant association with successful deployment and complications. A total of 2072 patients were included. VCDs were successfully deployed in 95.2% of the patients with 4.8% of perioperative complications, which included minor oozing from the puncture site, small hematoma less than or equal to 5 cm, large hematoma greater than 5 cm, pain, and loss of vascular access. Therapeutic (vascular interventional radiology (VIR) and neuro-interventional radiology (NIR)) procedures (OR 3.03, 95% CI 1.51-6.09, p = 0.002), use of Angioseal (OR 5.26, 95% CI 3.13-8.33), p < 0.001), and no use of antiplatelet medications (OR 0.47, 95% CI 0.22-0.97, p = 0.041) were independently associated with successful deployment of VCDs when controlled for other risk factors. Smoking (OR 3.50, 95% CI 2.00-6.05, p = <0.001), use of antiplatelet (OR 2.01, 95% CI 1.04-3.87, p = 0.037) and use of heparin (OR 1.78, 95% CI 1.10-2.86, p = 0.018) were independently associated with higher complication rates. VCD's were successfully deployed in 95.2% of the patients with 4.8% of perioperative minor complications.

General practitioner gender and use of diagnostic procedures: a French cross-sectional study in training practices

ObjectivesThe acceleration in the number of female doctors has led to questions about differences in how men and women practice medicine. The aim of this study was to assess the...

An automated approach to high-plex cytometric immunophenotyping with CyTOF XT

Abstract CyTOF® mass cytometry is a single-cell analysis platform that uses isotope-tagged antibodies to resolve 50-plus markers in a single tube without signal compensation, making CyTOF ideal for routine immunophenotyping. CyTOF XT™, the latest CyTOF system, features automated sample acquisition. Stained samples were acquired in parallel using the automated CyTOF XT system and manually, using the Helios™ system, to assess performance of the automated system. Multiple suspension mass cytometry staining workflows were evaluated. Population frequencies and resolution indices for markers were assessed by manual gating. There was no significant difference between population frequencies analyzed between the two CyTOF systems. On average, samples acquired on CyTOF XT resulted in greater resolution between positive and negative populations compared to Helios. The Maxpar® Direct™ Immune Profiling System, which comprises the Maxpar® Direct™ Immune Profiling Assay™ and Maxpar Pathsetter™ software, was also compared on the CyTOF XT and Helios systems. The Maxpar Direct Immune Profiling Assay includes a 30-marker panel in a dry, single-tube format for staining human whole blood or PBMC. Maxpar Pathsetter automates reporting of population statistics and stain assessments for the panel. Maxpar Pathsetter showed comparable population frequencies between the two CyTOF systems and improved staining assessment on CyTOF XT. Overall, these studies find that the CyTOF XT system generates better signal resolution than the Helios system. Automated acquisition by CyTOF XT enables researchers to accurately and reproducibly streamline human immunophenotyping. For Research Use Only. Not for use in diagnostic procedures.

Imaging Mass Cytometry identifies structural and cellular composition of the mouse tissue microenvironment

Abstract Imaging Mass Cytometry™ (IMC™) is a vital tool to deeply characterize the complexity and diversity of any tissue without disrupting spatial context. The Hyperion™ Imaging System utilizes IMC, based on CyTOF® technology, to assess up to 40 individual structural and functional markers in tissues, providing unprecedented insight into the organization and function of tissue microenvironment. We have previously demonstrated the application of IMC in combination with Maxpar® panel kits on human tissues. Here, we showcase Maxpar OnDemand Antibodies for IMC application including 11 new highly relevant markers to construe cellular and molecular composition of mouse tissues. We analyzed a normal mouse tissue microarray using IMC spatial proteomic analysis. Tissues were stained with a 20-marker panel designed to highlight tissue architecture and major immune lineage markers. We generated a detailed spatial map of the diverse tissue architecture and successfully identified immune, epithelial, and stromal cell populations in various mouse tissues. Additionally, we classified the activation state of immune cell populations, adhesion state of epithelial cells, and molecular composition of the extracellular matrix. This work demonstrates the capability of IMC to identify subcellular localization of cellular and structural markers in the mouse tissue microenvironment. Future studies utilizing IMC in combination with Maxpar OnDemand Antibodies will enable in-depth phenotypic characterization of the tissue microenvironment in various mouse models of development and disease, and thus provide the basis for the use of high-multiplex imaging in preclinical investigations. For Research Use Only. Not for use in diagnostic procedures.

Extending the capabilities of a high-parameter immunophenotyping assay with cytoplasmic staining applications for mass cytometry

Abstract Maxpar® Direct™ Immune Profiling Assay™ (Cat. No. 201325) is a 30-marker panel for suspension mass cytometry. This panel provides an unprecedented sample-to-answer solution for detecting and analyzing 30 surface markers in a single experiment. The 18 open mass channels in the Maxpar Direct Assay facilitate panel expansion and enable flexibility for higher multiplexity and applications. Among the potential complementary applications, intracellular cytokine staining (ICS) is of particular interest as it may be used to assess infiltrating immune cell phenotypes in the tumor microenvironment. However, for the purpose of assessing cell viability in this workflow, the effectiveness of the Cell-ID™ Intercalator-Rh (103Rh, Cat. No. 201103) included in the Maxpar Direct Assay is in question, as cell permeabilization during ICS can potentially damage the DNA-intercalator bond. In this study, we investigated the compatibility of 103Rh with intracellular staining. We stained either human peripheral blood mononuclear cell or whole blood samples with the Maxpar Direct Assay followed by intracellular staining for the detection of expressed cytokines. We demonstrate that 103Rh provides equivalent functionality as a cell viability indicator during intracellular staining for cytoplasmic proteins compared to the benchmark Cell-ID Cisplatin-194Pt (Cat. No. 201194). This work was designed to support use of the Maxpar Direct Immune Profiling Assay in combination with additional intracellular markers. Overall, these findings expand the applicability of Cell-ID Intercalator-Rh (103Rh) to processes that involve cytoplasmic staining. For Research Use Only. Not for use in diagnostic procedures.

Gasdynamic electron cyclotron ion sources: Basic physics, applications, and diagnostic techniques.

The gasdynamic electron cyclotron resonance (ECR) ion source is a type of the device in which the ionization efficiency is achieved primarily due to a high plasma density. Because of a high particle collision rate, the confinement is determined by a gasdynamic plasma outflow from a magnetic trap. Due to high efficiency of resonant heating, electrons gain energy significantly higher than that in inductively or capacitively coupled plasmas. As a consequence of such a parameter combination, the gasdynamic ECR plasma can be a unique source of low to medium charged ions, providing a high current and an ultimate quality of an ion beam. One of the most demanded directions of its application today is a development of high-current proton injectors for modern accelerators and neutron sources of different intensities. Special plasma parameters allow for the use of diagnostic techniques, traditional for multiply charged ECR plasmas as well as for other types of discharges with a high plasma density. Among the additional techniques, one can mention the methods of numerical simulation and reconstruction of the plasma density and temperature from the parameters of the extracted ion beams. Another point is that the high plasma density makes it possible to measure it from the Stark broadening of hydrogen lines by spectroscopy of plasma emission in the visible range, which is a fairly convenient non-invasive diagnostic method. The present paper discusses the main physical aspects of the gasdynamic ECR plasma, suitable diagnostic techniques, and possibilities and future prospects for its various applications.

Detection of minimal residual disease (MRD) in colorectal cancer (CRC) patients UICC stage II/III by ultra-deep sequencing of cfDNA from post-surgery plasma.

26 Background: Detection of primary tumor mutations in cell-free DNA (cfDNA) of post-surgery plasma of patients with R0-resected not-metastasized solid tumors is a strong indicator of recurrence of disease. We explored whether ultra-deep sequencing of cfDNA could improve sensitivity and specificity with respect to time-to-progression. Methods: 84 CRC patients UICC stage II/III were recruited into the prospective, observational study “Molecular Signatures in Colorectal Cancer”. Matched tumor tissue samples, plasma depleted blood cells (PDBC), and cfDNA (drawn 1 to 34 days after R0-resection, median 7 days) were processed with the Roche AVENIO Tumor Tissue and ctDNA Surveillance Kits*. Samples of 79 patients passed all quality controls, in particular cfDNA was sequenced ultra-deep with a median of 180 Mio. instead of 50 Mio. reads/sample. Somatic variants were identified with AVENIO Oncology Analysis software 2.0*. PDBC informed germline variants were removed. If a tissue baseline variant was detected in cfDNA with a significant adjusted p-value, the patient was defined ctDNA+, and ctDNA- otherwise. Results: 8 ctDNA+ patients (28 variants, median AF = 0.15%) were identified of which 4 had a progression of disease at two years. Sensitivity was 44% (95% CI [0.137, 0.788]), specificity was 94% (95% CI [0.86, 0.984]), positive predictive value was 50% (95% CI [0.157, 0.843]), and negative predictive value was 93% (95% CI [0.843, 0.977]). Comparison of time-to-progression of ctDNA+ and ctDNA- patients using the log-rank test resulted in a p-value of 0.0058. Comparison of survival times of ctDNA+ and ctDNA- patients resulted in a p-value of 0.0333. Multivariate analyses of times-to-progression resulted in ctDNA-status (p = 0.0022, hazard ratio (HR) = 7.098) and neoadjuvant therapy (p = 0.0010, HR = 6.618) as significant parameters. Conclusions: Even in this small cohort of CRC UICC stage II/III patients, MRD detection in post-surgery plasma is the strongest predictor of shorter time to progression. Ultra-deep sequencing of cfDNA samples did not influence MRD detection on a patient-level. *for Research Use Only; not for use in diagnostic procedures.

Analytical Validation of a New Immunoenzymatic Method for the Measurement of Feline Parathyroid Hormone in Cats with Chronic Kidney Disease.

Simple SummaryParathyroid hormone (PTH) is involved in many metabolic diseases, such as chronic kidney disease (CKD) and calcium disorders, and its measurement could be of clinical utility. However, available methods for the measurement of feline PTH are limited and not widely accessible. The aim of this study was to perform the analytical validation of a new method for PTH measurement in cats. Thirty-eight cats affected with CKD were included. The analytical protocol provided an evaluation of the precision, accuracy, and storage stability at different temperatures. The method investigated showed good precision and accuracy and good stability for 1 week of storage at freezing temperatures. The method was validated in cats, allowing its future use in diagnostic procedures.The determination of parathyroid hormone (PTH) in cats could be of clinical utility in many metabolic disorders, such as renal diseases, hypercalcemia, or nutritional imbalances. However, the available methods for the measurement of feline PTH are limited, not widely available, and need radioimmunoassays. The aim of this study was to perform the analytical validation of a new immunoenzymatic method for the measurement of feline PTH. Thirty-eight cats affected with chronic kidney disease (CKD) were included. PTH was measured using a two-site immunoenzymatic method validated in humans and dogs (ST AIA-PACK® Intact PTH, Tosoh Bioscience, Tessenderlo, Belgium). The analytical validation provided the evaluation of precision (intra-assay and inter-assay), accuracy (linearity under dilution (LUD) and spike recovery test (SRT)), and the storage stability of serum samples at 20 °C, 4 °C, and −20 °C. The method showed good precision (intra-assay CVs (coefficient of variations) 3.19–9.61%; inter-assay CVs 9.26–15.28%). In both the intra- and inter-assays, the highest imprecision was found with the low concentration pool (9.61% and 15.28%) and accuracy (LUD and SRT r2 = 0.99, p < 0.001), while the stability was optimal up until 7 days at −20 °C (−7.7%). The method was successfully validated in cats, allowing its future use in diagnostic procedures.

Agreements and controversies of national guidelines for bronchiolitis: Results from an Italian survey.

IntroductionSignificant variations in the management of bronchiolitis are often recorded, and, in parallel, to recommend a univocal clinical approach is challenging and still questioned. This study is aimed to evaluate the diagnostic and therapeutic management of bronchiolitis in children adopted by Italian pediatricians following the national guidelines.Material and MethodsA survey study was designed and carried out by sending an email an open‐ended questionnaire developed by an expert panel of the Scientific Board of the Italian Society of Pediatric Allergology and Immunology (SIAIP). Questions were designed according to the national intersociety consensus document on treatment and prevention of bronchiolitis in newborns and infants.ResultsOverall, 234 pediatricians were taking part in the study. When diagnosing bronchiolitis, only 44.01% (103/234) of participants correctly followed the national guidelines. All participants (100%) would perform laboratory tests and/or radiological exams. 44.01% administered oxygen (O2) when O2 saturation was minor than 92%. About the therapeutic regimen, marked discrepancies between national guidelines and recorded answers were reported. Indications for hospital admission and discharge criteria were in line with the national guidelines.ConclusionsThere is a significant practice variation in the management of acute bronchiolitis among Italian physicians. Some wrong attitudes need to be further discouraged, such use of diagnostic procedures and therapeutic approaches. Further research is urgently required to define the best management of patients with bronchiolitis and implement strategies to standardize care and improve the quality of care.

Adherence to guidelines of diagnostic and therapeutic practices for suspected infections of cardiac implantable electronic devices

Abstract Background Despite guidelines describing the optimal diagnostic and therapeutic procedures of patients with suspected infection of cardiac implantable electronic device (CIED), the management of such patients is often challenging. Purpose The aim of this study was to describe our diagnostic and therapeutic practices for suspected CIED infection and to compare them to European Heart Rhythm Association (EHRA) guidelines. Methods Patients hospitalized in our tertiary care hospital for suspected CIED infection from 2014 to 2019 were retrospectively included. We applied the EHRA classification and compared diagnostic and therapeutic management to EHRA guidelines. Results Among 184 patients (mean age 72.3±12.4 years), 137 had a proven infection of the lead (either by TTE/TEE, by [18F]FDG PET/CT, or by positive culture of the lead) or an isolated pocket infection without proof of lead infection and 47 had no proof of infection of CIED. According to EHRA classification, CIED infection was considered as definite in 145 patients, possible in 31 and excluded in 8. Regarding recommended diagnostic procedures, blood cultures were performed in 90.8%, TTE in 97.8%, TEE in 85.9%, [18F]FDG PET/CT in 50.5% and imaging for embolisms in 78.3% of the patients. Compared to therapeutic recommendations for the 145 cases of definite CIED infection, device removal was performed in 96 patients (66.2%), antibiotic therapy was prescribed in 130 (89.7%), with a duration equal or superior to the one recommended in 105 (72.4%) of the patients. One-year survival rate was 66.3% and was better in patients with device removal than in patients without, both in the whole population (respectively 77.7% and 48.6%) and in the patients with definite CIED infection (80.0% vs 49.0%). After adjustement on age and Charlson's score, patients without device removal had a significantly higher risk of 1-year mortality (HR=3.40 [1.84–6.26], p=0.0001). Conclusion Suspicion of CIED infection are difficult situations. The use of theoretical classification helps to define the different types of cases and the resultant therapeutic approach. In daily practice, our study shows that the strict adherence to these guidelines is not always possible. This study shows a relatively good use of diagnostic procedures. However, theoretical advice for medical therapy and removal procedures are not always properly followed. To improve patients' prognosis, infected device removal must be systematically discussed in multidisciplinary endocarditis team, weighing the benefit-risk ratio of the strict appliance of the guidelines. Funding Acknowledgement Type of funding sources: None. Diagnostic comparisonTherapeutic comparison

Systematic Undercoding of Diagnostic Procedures in National Inpatient Sample (NIS): A Threat to Validity Due to Surveillance Bias.

Health services research often relies on readily available data, originally collected for administrative purposes and used for public reporting and pay-for-performance initiatives. We examined the prevalence of underreporting of diagnostic procedures for acute myocardial infarction (AMI), deep venous thrombosis (DVT), and pulmonary embolism (PE), used for public reporting and pay-for-performance initiatives. We retrospectively identified procedures for AMI, DVT, and PE in the National Inpatient Sample (NIS) database between 2012 and 2016. From January 1, 2012, through September 30, 2015, the NIS used the International Classification of Diseases, Ninth Revision (ICD-9) coding scheme. From October 1, 2015, through December 31, 2016, the NIS used the International Classification of Diseases, Tenth Revision (ICD-10) coding scheme. We grouped the data by ICD code definitions (ICD-9 or ICD-10) to reflect these code changes and to prevent any confounding or misclassification. In addition, we used survey weighting to examine the utilization of venous duplex ultrasound scan for DVT, electrocardiogram (ECG) for AMI, and chest computed tomography (CT) scan, pulmonary angiography, echocardiography, and nuclear medicine ventilation/perfusion () scan for PE. In the ICD-9 period, by primary diagnosis, only 0.26% (n = 5930) of patients with reported AMI had an ECG. Just 2.13% (n = 7455) of patients with reported DVT had a peripheral vascular ultrasound scan. For patients with PE diagnosis, 1.92% (n = 12 885) had pulmonary angiography, 3.92% (n = 26 325) had CT scan, 5.31% (n = 35 645) had cardiac ultrasound scan, and 0.45% (n = 3025) had scan. In the ICD-10 period, by primary diagnosis, 0.04% (n = 345) of reported AMI events had an ECG and 0.91% (n = 920) of DVT events had a peripheral vascular ultrasound scan. For patients with PE diagnosis, 2.08% (n = 4805) had pulmonary angiography, 0.63% (n = 1460) had CT scan, 1.68% (n = 3890) had cardiac ultrasound scan, and 0.06% (n = 140) had scan. Small proportions of diagnostic procedures were observed for any diagnoses of AMI, DVT, or PE. Our findings question the validity of using NIS and other administrative databases for health services and outcomes research that rely on certain diagnostic procedures. Unfortunately, the NIS does not provide granular data that can control for differences in diagnostic procedure use, which can lead to surveillance bias. Researchers and policy makers must understand and acknowledge the limitations inherent in these databases, when used for pay-for-performance initiatives and hospital benchmarking.

Abstract 1307: Assessment of long IVT mRNA fragments with the Fragment Analyzer system

Abstract Quality control analysis of long in vitro transcribed (IVT) RNAs is challenging due to the lack of commercially available kits for sizing RNA over 6,000 nt, thus, to analyze long IVT RNAs, an extended separation method was developed for the Agilent 5200 Fragment Analyzer using a commercially available RNA ladder. IVT mRNA was produced using the RiboMAX Large Scale RNA Production System (Promega). RNA transcripts 9,000 and 10,000 nt in length were diluted in nuclease-free water and separated on the Agilent 5200 Fragment Analyzer system with the Agilent RNA kit (15 nt) (p/n DNF-471) with the following modifications. The Lonza RNA marker (Lonza) (long RNA Ladder) replaced the Agilent RNA Ladder and was diluted in nuclease-free water to 96 ng/μL. The long RNA Ladder was added to the Agilent RNA Diluent Marker (15 nt) (p/n DNF-369-0004) according to the RNA kit protocol. The standard separation method (8 kV for 45 minutes) for the RNA kit (15 nt) was manually altered to the extended RNA method (4 kV for 90 minutes) in the Agilent 5200 Fragment Analyzer software for fragments greater than 6,000 nt. The extended RNA method with the long RNA Ladder was employed for all subsequent runs. The long RNA Ladder separated with the extended method displayed enhanced resolution compared to separation with the standard method as seen by the increased spacing between the ladder peaks and the increased sharpness of the 9,000 nt peak. The extended RNA method reported a lower average sizing percent error (-0.7 %) over the entire concentration range of the kit compared to the standard RNA method (8.4 %) for the 9,000 nt sample. The 10,000 nt IVT mRNA average percent sizing error was similar between the two separation methods throughout the dilution series. Using the extended method, the 9,000 and 10,000 nt IVT mRNA samples were successfully analyzed with good sizing precision and accuracy, demonstrating that a commercially available RNA ladder can be used with the Fragment Analyzer system to provide accurate sizing of large IVT mRNA samples. The extended RNA method is recommended when extremely accurate sizing or high resolution is needed for determining the presence of degradation or sizing differences from incomplete transcription in IVT mRNA samples longer than 6,000 nt. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Kyle D. Luttgeharm, Solange Borg, Chava Pocernich. Assessment of long IVT mRNA fragments with the Fragment Analyzer system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1307.

Abstract 1676: A comprehensive whole blood CyTOF immune monitoring panel with expanded surface and intracellular markers using the Maxpar Direct Immune Profiling Assay

Abstract In-depth monitoring of the immune response to cancer and infection is vital to ascertain disease status and to assess immunotherapeutic options. Time-of-flight technology, the basis of CyTOF® mass cytometry, enables multiplex proteomic cellular phenotyping with 50 or more markers, making it ideal for comprehensive immune profiling. Unlike fluorescence-dependent approaches, which require signal compensation that makes the development of larger panels more challenging, CyTOF utilizes monoisotopic metal-tagged antibodies that exhibit minimal background signal, enabling the highest-resolution multiparametric landscape of a single cell. The Maxpar® Direct™ Immune Profiling Assay™ is a pre-titrated dried-down 30-antibody cocktail preparation for immune profiling of human whole blood or PBMC. This assay is used with Maxpar Pathsetter™ software, which resolves whole blood into 37 immune populations comprising major lineage populations and their subsets, such as CD4 Th subsets and B and T cell memory cells, as well as stratifications of myeloid populations. The resulting system is a simple sample-to-answer solution for immune monitoring studies. Here, we expanded the 30-marker assay with 14 additional antibodies comprising pertinent targets of immunotherapy, including the exhaustion markers PD-1, PD-L1, Tim-3 and CTLA-4, and co-stimulation markers 4-1BB and ICOS. We also demonstrated the compatibility of the assay with downstream intracellular staining for cytoplasmic markers IFN-γ, TNF-α, IL-2, perforin and granzyme B for assessment of cellular function in 4h PMA/ionomycin-stimulated whole blood cultures. Next, we modified the existing Pathsetter model to automate the analysis of whole blood stained with the expanded panel to generate reports on key immune cell populations, percentages of exhausted cells and cell subsets producing cytokines. Last, to demonstrate the ability of this expanded panel to identify antigen-specific T cell subsets accompanied by their in-depth phenotypic assessment, we tested this panel against CMV peptide-stimulated whole blood samples. We demonstrated the flexibility of the Maxpar Direct Immune Profiling Assay in panel customization and a streamlined workflow for automated analysis to enable comprehensive immune profiling of human whole blood. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Shariq Mujib, Stephen K. Li, Nick Zabinyakov, Christina Loh. A comprehensive whole blood CyTOF immune monitoring panel with expanded surface and intracellular markers using the Maxpar Direct Immune Profiling Assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1676.

Abstract 249: Comparison of variant classification between molecular diagnostics experts and decision support software

Abstract INTRODUCTION: Assessing the actionability of variants identified in next-generation sequencing (NGS) of solid tumors is challenging. Recommendations from AMP/ASCO/CAP/ACMG (Li et al. JMD 2017) provide guidelines for prioritizing variants based on clinical significance and actionability. Sirohi et al. (JMD 2020) reported on interrater agreement with the AMP guidelines by distributing 51 variants from solid tumor NGS to 20 participants at 10 US academic molecular diagnostics laboratories. Initial agreement based on independent assessment amongst the 20 experts was fair (Cohen κ = 0.35). To explore the potential for decision support software to streamline interpretation and improve the reliability of tier classification, we evaluated the same variants with NAVIFY® Mutation Profiler*, a CE-IVD somatic variant interpretation tool that automatically assigns tiers based on curated public evidence. METHODS: Reverse annotation from protein or coding sequence changes was completed with TransVar (Zhou et al. Nat Methods 2015) to obtain hg38 genomic coordinates for the 51 variants. Variants were prepared in VCF files and uploaded to NAVIFY Mutation Profiler (v2.0.0.22e0193) for analysis. The software assigned tier classifications using evidence in Roche curation content version 2.33.0 (released Nov 10, 2020). The default tier for each variant was compared to the tier assigned by the most experts in Sirohi et al. If there was a tie, the higher tier was used for comparison (e.g. Tier II when there was a tie between Tier II and III). RESULTS: There was good agreement (Cohen κ = 0.61) in tier assignments for 37 variants with curation content in NAVIFY Mutation Profiler compared to consensus expert curation in Sirohi et al. Most disagreements could be explained by emerging biomedical evidence, such as phase II clinical trial results for NFE2L2 in squamous cell lung carcinoma, or recommendations in NCCN guidelines. For example, ERBB2 mutations were up-classified by NAVIFY Mutation Profiler to Tier I due to NCCN therapy recommendations, including trastuzumab in combination with lapatinib or pertuzumab for ERBB2-amplified colorectal cancer and trastuzumab emtansine for non-small cell lung cancer with ERBB2 exon 20 insertions. Of the 14 variants without curation in the database, none were actionable, and the experts had classified 11 as Tier III and 3 as Tier II. CONCLUSIONS: When curation content was available, there was high agreement between the assigned tier in NAVIFY Mutation Profiler and the most common tier assigned by 20 molecular diagnostics experts in a previously published study. The continuous growth in new biomedical evidence requires staying current with the latest guidelines and clinical trial results, or the use of regularly updated databases, to accurately interpret somatic cancer variants from NGS test results. * This product is for Research Use Only; Not for use in diagnostic procedures in the US. Citation Format: Stephanie J. Yaung, Shuba Krishna, Sidney Scudder, Maximilian Schmid, John F. Palma. Comparison of variant classification between molecular diagnostics experts and decision support software [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 249.

Novel Multiplexed Assays for Antibody Isotypes against SARS-CoV-2 Using Flow Cytometry

Abstract The ongoing COVID-19 pandemic has prompted intense research on the role of antibodies in characterizing the emergence and sustenance of the immune response. Antibody isotype information is important in understanding disease mechanism, seroconversion, and vaccine response. While several methods provide qualitative information on SARS-CoV-2 antibodies, there is a need for quick methods with high detection sensitivity that provide relative response levels against multiple SARS-CoV-2 antigens. In this study, we show results from the development of a high-sensitivity bead-based immunoassay for flow cytometry that enables detection of IgG, IgM, or IgA antibodies in serum or plasma samples against multiple SARS-CoV-2 antigens. Antigenic targets include SARS-CoV-2 nucleoprotein, S1-subunit of the spike protein, and receptor-binding domain of the spike protein. The assay allows for quick processing, displays clear visual shifts, and provides fluorescent intensity information allowing for high detection sensitivity. Application of the assay to 30 PCR positive SARS-CoV-2 samples and 30 pre-pandemic samples provides high sensitivity and specificity for IgG analysis. IgG detection was observed in SARS-CoV-2 samples ranging from 6–75 days post-onset of symptoms in the study. IgM and IgA antibodies were also detected in several samples. The assay demonstrates good precision, with low %CVs of &amp;lt;10% typically observed. Given that SARS-CoV-2 research encompasses both cellular and serological aspects, the ability to obtain antibody isotype information on flow cytometry platforms that enable cellular as well as bead-analysis can benefit many researchers. For Research Use Only. Not for use in diagnostic procedures.

A novel grey scale and Power Doppler ultrasonographic score for idiopathic inflammatory myopathies: Siena Myositis Ultrasound Grading Scale.

No clear-cut guidelines exist on the use of diagnostic procedures for idiopathic inflammatory myopathies (IIM) and only minimal and conflicting data report the use of ultrasound (US). In this regard, we aimed to assess if grey-scale (GS) and Power Doppler (PD) US, graded with a 0-3-point scale, may be a reliable tool in a cohort of patients affected by IIM. All patients underwent US examination of both thighs in axial and longitudinal scans. Oedema and atrophy, both assessed in GS and PD, were graded with a 0-3-point scale. Spearman's test was used to identify the correlations between US and clinical and serological variables. A total of 20 patients were included. Six and two patients were evaluated twice and three times, respectively. Muscle oedema was found to be directly correlated with physician global assessment (PhGA), serum myoglobin and PD and negatively with disease duration. PD score was positively correlated to PhGA and negatively to disease duration. Muscle atrophy directly correlated with Myositis Damage Index, disease duration and patient's age. The single-thigh sub-analysis evidenced a direct correlation between PD score and Manual Muscle Test. In our cohort, we found that oedema and PD are strictly related to early, active myositis, suggesting that an inflamed muscle should appear swollen, thickened and with Doppler signal. Conversely, muscle atrophy reflects the age of the patient and the overall severity of the disease. Such findings shed a new, promising, light on the role of US in diagnosis and monitoring of IIMs.

Abstract PS2-03: Comparison of pathologist reads of sp142 and sp263 with quantitative measurement of protein and mRNA in triple negative breast cancer

Abstract Background: PD-L1 SP142 immunohistochemistry (IHC) assay has been approved as a companion test by the US Food and Drug Administration (FDA) to identify eligibility for atezolizumab therapy in patients with advanced triple negative breast cancer (TNBC) but a number of studies suggest the assay suffers from poor reproducibility. Using readings of 70 TNBC chromogenic FDA approved assays from 19 pathologists in a previous study as a baseline, we compared pathologist reads to quantitatively measured mRNA and protein expression Methods: Formalin-fixed paraffin-embedded (FFPE) slides representing primary invasive triple negative breast cancer (stage I-III) from 100 patients between 2012-16 were selected from the Yale Pathology archives. Slides were macrodissected to tumor enrichment for quantitative assessment of CD274 (PD-L1 mRNA) measured using a closed-system, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) research use only (RUO)* prototype assay on the GeneXpert® instrument. We also measured protein expression levels using the AQUA method of quantitative immunofluorescence (QIF) in both the tumor and non-tumor compartments on full sections by QIF stained using SP142 in a lab derived test (LDT). The IHC stained slides were prepared using SP142 and SP263 assays prepared exactly according the FDA approved label followed by reading by 19 pathologists. This study was approved by Yale Human Investigation IRB protocol ID 9505008219.Results: Previous work from our group showed overall percent agreement for both the SP142 and SP263 IHC assays read by pathologists was in the 40-50% range. We used the median CD274 score to compare positive (IC≥1) vs negative (IC&amp;lt;1) and found that the levels of mRNA were not statistically significantly different between the two categorical scores. However, quantitative measurement of protein expression (including both tumor and non-tumor regions) showed statistically significant differences between pathologist read PD-L1 positive/negative scoring for SP142 (p=0.0004) and SP263 (p=0.0185). Concordance of quantitative PD-L1 measurement between protein QIF scores and transcript RT-qPCR levels was modest, with a Spearman coefficient r = 0.18. Conclusions: By chromogenic IHC, using the FDA approved assays, pathologist read scoring shows no difference to mRNA for CD274. Quantitative continuous scoring of protein expression show that, on average, when pathologists score IC≥1, there is more protein present than when they score IC&amp;lt;1. Further studies are needed to determine if RNA and protein for PD-L1 are concordant and to determine which assay(s) and cutoff values are best correlated with clinical outcomes on atezolizumab therapy. *For Research Use Only - Not for use in diagnostic procedures. Not approved or reviewed by any regulatory body Citation Format: Swati Gupta, Vesal Yaghoobi, Aileen Fernandez, Leena McCann, Jodi Weidler, Michael Bates, Emily Reisenbichler, David Rimm. Comparison of pathologist reads of sp142 and sp263 with quantitative measurement of protein and mRNA in triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-03.

Abstract PS4-03: An automated DNA methylation assay for monitoring treatment response in patients with metastatic breast cancer

Abstract BACKGROUND: Previously, we demonstrated the clinical validity of cMethDNA, a circulating methylated tumor DNA (ctDNA) assay, in serum samples from TBCRC 005 (J Clin Oncol, 2017; 35:751-758) to predict progression free- and overall-survival, and to monitor therapeutic response in patients with stage IV breast cancer. Here, Johns Hopkins (JH) and Cepheid partnered to develop an automated GeneXpert (GX) cartridge-based system to provide quantitative measures of DNA methylation within 5 hours.METHODS: With a goal of discriminating stage IV breast cancer from healthy and benign breast disease with high sensitivity and specificity, we evaluated breast cancer-specific DNA methylation markers (selected through comprehensive methylome analysis) in STRECK tube plasma of 46 patients with metastatic breast cancer enrolled in Individualized Molecular Analyses Guide Efforts in Breast Cancer (IMAGE II trial), 17 benign breast disease and 9 healthy normal controls (J0888 repository). Blood from IMAGE II participants was collected upon disease progression. A newly designed GX Breast Cancer Monitoring Assay for research use only (RUO*) first converted unmethylated CpG sites in ctDNA from 1 ml plasma with bisulfite. The sample was then split into two methylation detection cartridges, which quantitated DNA methylation of 9 markers along with an ACTB reference. Cumulative methylation (CM) of the 9-gene panel was calculated using a novel algorithm. Performance was assessed based on Receiver Operating Characteristic (ROC) curves and Mann-Whitney analyses.RESULTS: The GX Breast Cancer Monitoring Assay (RUO)* showed that the 9-gene panel was significantly more methylated in cancer compared to normal/benign plasma samples (median for cancer: 428.0 CM units versus for benign: 0.0 CM units; P&amp;lt; 0.0001), and revealed a sensitivity of 85% and specificity of 92%, using a cumulative methylation threshold of 35.5 units based on ROC area under the curve (AUC) = 0.909 (95% CI 0.836 – 0.982, P&amp;lt;0.0001). We will present comparisons of the GX results to cMethDNA, the gold standard assay, which reported 85-90% sensitivity at 90% specificity.CONCLUSIONS: We identified a panel of methylated DNA markers that discriminates stage IV breast from benign breast disease and healthy normal subjects using ctDNA. Our automated cartridge-based assay prototype demonstrates high sensitivity and specificity for detecting invasive breast cancer. Its ability to assess changes in DNA methylation will be tested next with clinical trial samples collected longitudinally during treatment. This assay has potential clinical utility in monitoring therapeutic response and predicting disease recurrence.* For Research Use Only. Not for use in diagnostic procedures. Not reviewed by any regulatory body. Citation Format: Mary Jo Fackler, Suzana Tulac, Neesha Venkatesan, Adam J. Aslam, Leslie M. Cope, Jennifer Lehman, Rita Denbow, Jeffrey Reynolds, Morgan Buckley, Bradley M. Downs, Kala Visvanathan, Christopher B. Umbricht, Antonio C. Wolff, Vered Stearns, Edwin W. Lai, Saraswati Sukumar. An automated DNA methylation assay for monitoring treatment response in patients with metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-03.

Metformin Therapy During Surgical Interventions andIodinatedContrast Agent Use

Metformin as first-line treatment in type 2 diabetes mellitus (T2 D) shows benefits in terms of reducing cardiovascular events, but the risk of a lactic acidosis as a serious adverse event especially in patients with decreased renal function is still relevant. Since the perioperative management of Metformin or its use in diagnostic procedures with contrast agents is inconsistent in literature and different in practice, the results of various guidelines are reviewed below showing the current state of evidence. Despite many guidelines, the evidence on both issues is low, as they are mainly based on consensus recommendations. The guidelines are still based on weak data and many international recommendations have clearly different statements. A fundamental problem with drugs is that expert information does specify eGFR limits for dose reduction, but not the method to be used. Depending on the formula, this can then lead to different treatment decisions. At present, it is not possible to give reliable recommendations for practice with the aim of minimising the interruption of therapy. For this reason, only a strictly conservative approach with 48-hour breaks before and after both measures can be recommended at present. For the situations mentioned in this overview, the question of the right approach has not yet been conclusively and definitely answered, therefore further studies should be carried out.